Structure, function, and disease biology of MICU1/MICU2

MICU1/MICU2的结构、功能和疾病生物学

基本信息

批准号：
10197754
负责人：
Zenon Grabarek
金额：
$ 45.03万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10197754
关键词：
Affinity Alleles Animals Binding Bioenergetics Biology Biophysics Calcium Calcium Binding Calcium Channel Calcium Signaling Calcium-Binding Proteins Cell Line Cells Clinical Communication Complex Cryoelectron Microscopy Data Defect Disease EF Hand Motifs Electrons Electrophysiology (science)Energy Metabolism Engineering Enzymes Escherichia coli Exercise Fatigue Fiber Genetic Genomics Goals Grant Histocytochemistry Histology Homeostasis Homologous Gene Human Human Genome Inherited Ion Channel Kinetics Knock-out Knockout Mice Lead Length Lesion Measures Membrane Potentials Metabolic Mitochondria Mitochondrial Myopathies Modeling Molecular Mus Muscle Muscle Contraction Muscle Fatigue Muscle Weakness Mutation Myopathy NADH Names Neurologic Organelles Oxygen Consumption Patients Performance Peripheral Physiology Plasma Positioning Attribute Process Production Property Proteins Regulation Reporting Resolution Rest Roentgen Rays Role Signal Transduction Skeletal Muscle Strenuous Exercise Structural Models Structure System Technology Testing Tissues Work X-Ray Crystallography common symptom design exercise intolerance genome editing in vivo loss of function mutation metabolomics mouse model mutant nervous system disorder neurotransmission response uptake

项目摘要

ABSTRACT Mitochondria uptake calcium via a calcium activated channel called the uniporter. Calcium uptake allows the organelle's metabolic state to be matched to rapidly changing energy requirements, and, in turn, tune key processes such as neurotransmission and muscle contraction. The uniporter is calcium-activated calcium channel regulated by the calcium binding heterodimer MICU1/MICU2. Mutations in MICU1 have recently been identified as a cause of a new form of a myopathy characterized by fatigue and exercise intolerance without the classical features of mitochondrial myopathy. The precise mechanisms by which MICU1 and MICU2 sense calcium to regulate the uniporter, and how lesions in this heterodimer lead to this highly unusual myopathy are not known. Through this dual PI grant we propose to: (1) Characterize the physicochemical properties of MICU1 and MICU2. The wild type proteins and mutants expressed in E. coli will be characterized with respect to the oligomeric state, structural stability, Ca2+ and Mg2+ binding affinities, and pH sensitivity. (2) Determine the structural basis for the regulation of the uniporter by MICU1 and MICU2. High resolution X-ray structures of MICU2 alone and of the MICU1/2 heterodimer in the apo and Ca2+-bound forms will be determined. Electron cryo-microscopy will be used to determine the structure of a native-like oligomer of MICU1/2 complex. (3) Investigate mitochondrial calcium dynamics in cellular systems. Using genome-editing technology we have engineered a powerful in vivo system of knockout cell lines for studying the effects of engineered and naturally occurring human mutations in MICU1 and MICU2 on the mitochondrial calcium transport kinetics and energetics, and (4) Understand the metabolic and bioenergetic basis of human MICU1 myopathy by investigating the Micu1-/- mouse as a model. We will characterize the muscle histology, mitochondrial bioenergetics, exercise performance and metabolomics, and single fiber contractility to test the hypothesis that loss of MICU1 leads to a myopathy by causing disturbances in energy metabolism. Our aims – spanning the molecular to animal physiology -- will yield a holistic and mechanistic understanding of the regulation of mitochondrial calcium uptake by MICU1 and MICU2 and its contribution to a newly described human myopathy.

抽象的线粒体吸收钙通过钙激活通道称为Uniter。钙吸收允许 Organelle的代谢状态与能源需求迅速变化相匹配，然后调整神经传递和肌肉收缩等关键过程。 UNITER是钙激活的钙由钙结合异二聚体MICU1/MICU2调节的通道。 MICU1中的突变最近已经被确定为一种新形式的肌病的原因线粒体肌病的经典特征。 MICU1和MICU2感知的确切机制钙调节静电蛋白的钙，以及该异二聚体中的病变如何导致这种非常不寻常的肌病不知道。通过此双PI赠款，我们建议：（1）表征的物理特性 MICU1和MICU2。大肠杆菌中表达的野生型蛋白质和突变体将以尊重的态度进行表征对于寡聚状态，结构稳定性，Ca2+和Mg2+结合亲和力以及pH敏感性。（2）确定通过MICU1和MICU2调节UNITER的结构基础。高分辨率的X射线结构将确定APO和Ca2+粘结形式中的MICU2和MICU1/2异二聚体的。电子冷冻微观镜将用于确定MICU1/2的天然样寡聚物的结构复杂的。（3）研究细胞系统中的线粒体钙动力学。使用基因组编辑技术我们已经设计了一个强大的在敲除细胞系的体内系统，用于研究工程和线粒体钙转运动力学和MICU2中的自然发生的人类突变能量学和（4）了解人类MICU1肌病的代谢和生物能基础将MICU1 - / - 小鼠作为模型进行研究。我们将表征肌肉组织学，线粒体生物能学，运动性能和代谢组学以及单纤维收缩性，以检验以下假设 MICU1的丧失通过引起能量代谢障碍而导致肌病。我们的目标 - 跨越分子到动物生理 - 将对调节产生整体和机械的理解 MICU1和MICU2的线粒体钙摄取及其对新描述的人的贡献肌病。